Over the past two years we have developed reliable methods for the fusion of human lymphocytes with a human plasma cell line to produce human x human hybridomas that synthesize monoclonal immunoglobulin. In preliminary studies we have prepared human hybridomas by fusion of the GM4672 plasma cell line with lymphoyctes isolated from a patient with immune thrombocytopenia and systemic lupus erythematosus. Antiplatelet antibodies synthesized by hybrid cells were detected using a solid phase ELISA system. Hybrids producing platelet-binding antibodies were cloned and 37 subclones obtained. One clone, HF2 1/17, produces an IgM K antibody that binds to platelets. Fab fragments of the HF2 1/17 antibodies had platelet-binding activity. In a competition ELISA system ristocetin inhibited antibody-platelet binding. In contrast, ADP, fibrinogen, epinephrine, thrombin and collagen did not inhibit. HF2 1/17 antibodies inhibited platelet aggregation induced by ristocetin. The proposed study includes the structural characterization and comparison of monoclonal autoantibodies which bind to platelets. This characterization will include sequencing of the NH2-terminal portion of the variable region and determination of immunoglobulin chain, class and isotype. Analysis of autoantibodies derived from a single patient and those from populations of patients with immune thrombocytopenia should reveal whether any particular structural form of immunoglobulin is associated with autoantibodies. Comparison of the framework regions and the hypervariable regions (complementarity determining regions) will allow conclusions about the structural similarities of autoantibodies. Identification of the antigenic determinant(s) on the platelet against which these antibodies are directed will receive considerable attention. These studies will include Western blotting of platelet membranes with antibody, analysis of platelet aggregation, and interaction of antibodies with mutant platelets. In addition, the effects of the autoantibody on platelet function will be determined. These monoclonal autoantibodies have potential for the detailed description of autoantibodies associated with immune thrombocytopenia.